2D Correlation Experiments:
HSQC, HMQC, HMBC,
BCMB/CHEM 8190
Two Dimensional NMR Spectra
A General Scheme: other mixing and evolution
periods can be added to increase dimensions
Preparation
Evolution 1
(Increment t1)
Mixing
Evolution 2
(observe t2)
time
Example: COSY – mixing is scalar coupling
90x
d1 (recover)
90x (mix)
t1 (evolve)
t2 (observe)
2D –NMR - FIDs are Transformed in t2, then in t1
t1
t1
FT
ν1
ν2
ν2
Heteronuclear Single Quantum Coherence
HSQC
1H-15N
Pairs in Amide Bonds of Proteins
Provide Convenient Reporters at Each
Amino Acid Along the Backbone Just
Requires 15N Labeling
HSQC Spectrum of H-N Amides in a Protein
Red – without PI(4)P
Blue – with PI(4)P
Phosphoinositide interactions with PH domain of FAPP1 at a bicelle surface
Heteronuclear Magnetization Transfer as Used
in the INEPT and HSQC Experiments
1H
1H(I)
90x
τ
15N
τ
180y
90y
15N(S)
90x
Iz Æ -Iy Æ -2IxSz Æ -2IzSy
τ = 1/4J
Evolution and Detection in HSQC
180x
180x
90x
t2
t1/2
t1/2
Sz (ωS, t1) (180 decouples I)
-2IzSy
90x
τ
180x
τ
decouple
-2IzSy cos(ωS t1) + 2IzSx sin(ωS t1)
Ix(π/2)+Sx(π/2)
2IySz cos(ωS t1) - 2IySx sin(ωSt1) (unobserved 2Q)
IzSz (J2τ) (chem shift refocuses)
-Ix cos(ωS t1)
Iz (ωI, t2)
-Ix cos(ωS t1)cos(ωI t2) -Iy cos(ωS t1)sin(ωI t2)
Obtaining Quadrature: Substitute a 90y 15N Pulse
and Save Separately (States)
)
1H(I
90x
15N(S)
τ
τ
180y
90y
90y
τ = 1/4J
Iz Æ -Iy Æ -2IxSz Æ 2IzSx
180x
180x
90x
t2
t1/2
t1/2
90x
τ
180x
τ
decouple
Sz (ωS, t1) (180 decouples I)
2IzSx
2IzSx cos(ωS t1) - 2IzSy sin(ωS t1)
Important Features of HSQC Spectra
• Start with 1H magnetization: gain γH / γN
A factor of 10 in sensitivity
• Detect 1H magnetization: gain (γH / γN)2
Another factor of 100
• Even though you must acquire 32-64 t1
points, far more sensitive than direct
detection.
1H-13C
HSQC can be
run at Natural
Abundance
2
3
1
Spectral Display of HMQC Looks Just Like HSQC
1H(I)
τ
τ
t2
180x
180x
t1/2
t1/2
90x 180y
15N(S)
90y
τ
90x
τ decouple
180x
Iz Æ -Iy Æ -2IxSz Æ -2IxSx
-2IxSx = (-IxSx + IySy) + (-IxSx –IySy) = zero and 2Q coherence
180x changes sign of IySy parts, hence interchanges zero and 2Q
Evolves as chemical shift of 15N, just like HSQC
Differences in relaxation properties and lack of direct coupling
HMBC – coherence transfers can be tuned to eliminate
one-bond coupling and emphasize long-range couplings
1H(I)
τ
τ
t2
180x
180x
t1/2
t1/2
90x 180y
15N(S)
90y
τ
90x
τ decouple
180x
Choose τ = 1/4JIS1 for three-bond: Iz Æ -Iy Æ -2IxSz Æ -2IxSx
-2IxSx = (-IxSx + IySy) + (-IxSx –IySy) = zero and 2Q coherence
Choose τ = (2n+1)1/2JIS1 for one-bond: -Iy Æ Iy Æ Iz = no signal
HMBC of Carbohydrates is Useful in
Identifying Linkages
HSQC
2
HMBC
3
1